It is generally recognized that reduced delivery of oxides of nitrogen in the smoke of tobacco products is desirable. Therefore, a number of methods have been developed to reduce the levels of nitrogen oxide precursors, such as nitrates, in smoking products. While some of these methods are based on ion exchange or crystallization mechanisms, others employ microbiological means for reducing the levels of certain nitrogen-containing compounds in tobacco materials.
The microbial processes and microorganisms employed may be either aerobic or anaerobic and may utilize dissimilatory or assimilatory pathways to metabolize the nitrogen-containing compounds. These processes and organisms, for example, include those of U.S. Pat. No. 3,747,608, British patent specification No. 1,557,253 (stated to be based on U.S. patent application No. 883,449, filed Mar. 6, 1978), UK patent applications No. 2,014,031A (based on Luxembourg application No. 79039, filed Feb. 9, 1978), 2,023,995A (stated to be based on U.S. patent application No. 916,323, filed June 15, 1978), Canadian Pat. No. 1,081,076 (based on Luxembourg application No. 77272, filed May 6, 1977, and Luxembourg application No. 77872, filed July 29, 1977), European patent application No. 0,005,082 (based on U.S. patent application No. 900,044, filed Apr. 25, 1978) and West German patent application No. P3100715.5, filed Jan. 13, 1981.
While some of these processes make use of bacteria that belong to the indigenous microflora of tobacco, each employs only non-thermophilic microorganisms as the active microbial agent. Each also employs only low temperature fermentation conditions--5.degree.-40.degree. C. For example, British patent specification No. 1,557,253 employs 5.degree.-35.degree. C., Canadian Pat. No. 1,081,076--25.degree.-35.degree. C., UK patent application No. 2,014,031A--25.degree.-35.degree. C., UK patent application No. 2,023,995A--20.degree.-40.degree. C., UK patent application No. 2,028,628A--5.degree.-37.degree. C., European patent application No. 0,005,082--30.degree.-40.degree. C., West German patent application No. P3100715.5--30.degree. C. and U.S. Pat. No. 3,747,608--24.degree.-40.degree. C.
Most of these processes also require that the tobacco materials be terminally sterilized (e.g., 121.degree. C. for 15 min at 15 psig) before contact with the microorganisms and that the fermentation be conducted under substantially aseptic conditions. The various anaerobic processes also usually require sparging of the fermentation broth with inert gases or other treatments to limit the oxygen concentration.
A number of these processes also require various additives to be incorporated into the fermentation broths or to supplement the tobacco material isolated from those broths after fermentation. For example, British patent specification No. 1,557,253 requires various organic compounds to be added to the tobacco materials, Canadian Pat. No. 1,081,076 and UK patent application No. 2,014,031A require D-glucose and other additives and West German patent application No. P3100715.5 requires that sugars be added to the broth. Plainly, any requirement for such additives increases the cost of such processes and may result in non-tobacco compounds being incorporated into the tobacco materials.
Other microbial-based processes for treating tobacco are also known in the art. For example, U.S. Pat. Nos. 2,000,855, 3,747,608 and 4,037,609 purport to describe microbial processes and microorganisms for degrading nicotine that may be present in tobacco. These processes, although again perhaps making use of bacteria that belong to the indigenous microflora of tobacco, are also non-thermophilic and employ low temperature fermentation conditions, e.g., 24.degree.-40.degree. C. (U.S. Pat. No. 3,747,608), 20.degree.-45.degree. C. (U.S. Pat. No. 4,037,609) and 30.degree.-40.degree. C. (U.S. Pat. No. 2,000,855).
In addition, Japanese Pat. No. 73 49,999 (C.A. 79:123942x), S. A. Ghabrial, "Studies On The Microflora Of Air-Cured Burley Tobacco", Tobacco Science, pp. 80-82 (1976), W. O. Atkinson et al., Ky. Agr. Exp. Sta. Lexington Ann. Report, 86, p. 22 (1973), A. Koiwai et al., "Fermentation of Tobacco, II. Variations In Fermentation Procedure And Its Effect On Total Particulate Matter And Benzo(a) pyrene", Tob. Sci, 15, pp. 41-3 (1971), and U.S. Pat. No. 2,317,792 purport to describe other microbial-based fermentation and curing processes for tobacco. Again, each of these processes employs non-thermophilic organisms and low temperature fermentation conditions, e.g., 25.degree.-50.degree. C. (Japanese Pat. No. 73 49,999), 30.degree.-35.degree. C. (S. A. Ghabrial) and 30.degree.-40.degree. C. (A. Koiwai et al.).
The growth of thermophilic microorganisms on "sweating" tobacco is known to occur. However, such organisms have not been employed to reduce the content of nitrogen-containing compounds in tobacco. Rather, they have only been described to affect the aroma and mildness of cigar tobacco. Such processes include, for example, those of C. F. English et al., "Isolation Of Thermophiles From Broadleaf Tobacco And Effect Of Pure Culture Inoculation On Cigar Aroma And Mildness", Applied Microbiol., 15, pp. 117-19 (January 1967), and B. Dumery and J. P. Albo, "Participation of Microorganisms In The Fermentation Of Dark Tobacco Submitted To A "Pre-Storage-Thermic Treatment Storage" Type Of Process", A du Tabac, Sect. 2-16, Bergerac, S.E.I.T.A. (1979-80).
In U.S. patent application No. 307,602, filed Oct. 1, 1981, a process is described for the dentrification of tobacco materials by the action of thermophilic microorganisms in high temperature fermentation processes. Specifically, the levels of nitrates and other nitrogen-containing compounds present in tobacco materials are reduced via an anaerobic dissimilatory metabolic pathway of thermophilic organisms. The process advantageously permits such reduction to be effected without the need for additives to the fermentation broth or tobacco materials.
As with any microbial-based process, the start-up of microbial activity is of importance to the overall process itself. It is generally recognized by those skilled in the art of fermentation that microbial enzymatic reactions are highly sensitive to forces of a mechanical nature, as well as temperature and pH conditions. Just as these factors are critical to a given microbial denitrification process, such as that disclosed in U.S. patent application No. 307,602, they are also important in the start-up of the microbial denitrification activity. A well-defined, easily controlled start-up process contributes to optimal microbial activity in the denitrification process. Though the denitrification process may be maintained almost indefinitely once it starts, the overall efficiency and commercial success of the process is increased by predictable and reliable means of start-up. Optimal output of denitrified products is also ensured when the denitrification process can be initiated via relatively short and efficient start-up procedures, or suspended in a similarly efficient manner, when required.